Development, Characterization, and Radiation Dosimetry Studies of 18F-BMS-986229, a 18F-Labeled PD-L1 Macrocyclic Peptide PET Tracer.

PURPOSE: In cancer immunotherapy, the blockade of the interaction between programmed death-1 and its ligand (PD-1:PD-L1) has proven to be one of the most promising strategies. However, as mechanisms of resistance to PD-1/PD-L1 inhibition include variability in tumor cell PD-L1 expression in addition to standard tumor biopsy PD-L1 immunohistochemistry (IHC), a comprehensive and quantitative approach for measuring PD-L1 expression is required. Herein, we report the development and characterization of an 18F-PD-L1-binding macrocyclic peptide as a PET tracer for the comprehensive evaluation of tumor PD-L1 expression in cancer patients. PROCEDURES: 18F-BMS-986229 was characterized for PD-L1 expression assessment by autoradiography or PET imaging. 18F-BMS-986229 was utilized to evaluate tumor PD-L1 target engagement in competition with a macrocyclic peptide inhibitor of PD-L1 (BMS-986189) over a range of doses using PET imaging. A whole-body radiation dosimetry study of 18F-BMS-986229 in healthy non-human primates (NHPs) was performed. RESULTS: In vitro autoradiography showed an 8:1 binding ratio in L2987(PD-L1 +) vs. HT-29 (PD-L1-) tumors, more than 90% of which could be blocked with 1 nM of BMS-986189. Ex vivo autoradiography showed that 18F-BMS-986229 detection was penetrant over a series of sections spanning the entire L2987 tumor. In vivo PET imaging in mice demonstrated a 5:1 tracer uptake ratio (at 90-100 min after tracer administration) in L2987 vs. HT-29 tumors and demonstrated 83%-93% specific binding of BMS-986189 within those dose ranges. In a healthy NHP dosimetry study, the resultant whole-body effective dose was 0.025 mSv/MBq. CONCLUSION: 18F-BMS-986229 has been preclinically characterized and exhibits high target specificity, low background uptake, and a short blood half-life supportive of same day imaging in the clinic. As the PET tracer, 18F-BMS-986229 shows promise in the quantification of PD-L1 expression, and its use in monitoring longitudinal changes in patients may provide insights into PD-1:PD-L1 immuno-therapy treatment outcomes.

Adjuvant Therapy of Nivolumab Combined With Ipilimumab Versus Nivolumab Alone in Patients With Resected Stage IIIB-D or Stage IV Melanoma (CheckMate 915).

PURPOSE: Ipilimumab and nivolumab have each shown treatment benefit for high-risk resected melanoma. The phase III CheckMate 915 trial evaluated adjuvant nivolumab plus ipilimumab versus nivolumab alone in patients with resected stage IIIB-D or IV melanoma. PATIENTS AND METHODS: In this randomized, double-blind, phase III trial, 1,833 patients received nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks (916 patients) or nivolumab 480 mg once every 4 weeks (917 patients) for ≤ 1 year. After random assignment, patients were stratified by tumor programmed death ligand 1 (PD-L1) expression and stage. Dual primary end points were recurrence-free survival (RFS) in randomly assigned patients and in the tumor PD-L1 expression-level < 1% subgroup. RESULTS: At a minimum follow-up of approximately 23.7 months, there was no significant difference between treatment groups for RFS in the all-randomly assigned patient population (hazard ratio, 0.92; 95% CI, 0.77 to 1.09; P = .269) or in patients with PD-L1 expression < 1% (hazard ratio, 0.91; 95% CI, 0.73 to 1.14). In all patients, 24-month RFS rates were 64.6% (combination) and 63.2% (nivolumab). Treatment-related grade 3 or 4 adverse events were reported in 32.6% of patients in the combination group and 12.8% in the nivolumab group. Treatment-related deaths were reported in 0.4% of patients in the combination group and in no nivolumab-treated patients. CONCLUSION: Nivolumab 240 mg once every 2 weeks plus ipilimumab 1 mg/kg once every 6 weeks did not improve RFS versus nivolumab 480 mg once every 4 weeks in patients with stage IIIB-D or stage IV melanoma. Nivolumab showed efficacy consistent with previous adjuvant studies in a population resembling current practice using American Joint Committee on Cancer eighth edition, reaffirming nivolumab as a standard of care for melanoma adjuvant treatment.

A multi-omic single cell sequencing approach to develop a CD8 T cell specific gene signature for anti-PD1 response in solid tumors.

Immune checkpoint blockade (ICB) has led to durable clinical responses in multiple cancer types. However, biomarkers that identify which patients are most likely to respond to ICB are not well defined. Many putative biomarkers developed from a small number of samples often fail to maintain their predictive status in larger validation cohorts. We show across multiple human malignancies and syngeneic murine tumor models that neither pretreatment T cell receptor (TCR) clonality nor changes in clonality after ICB correlate with response. Dissection of tumor infiltrating lymphocytes pre- and post-ICB by paired single-cell RNA sequencing and single-cell TCR sequencing reveals conserved and distinct transcriptomic features in expanded TCR clonotypes between anti-PD1 responder and nonresponder murine tumor models. Overall, our results indicate a productive anti-tumor response is agnostic of TCR clonal expansion. Further, we used single-cell transcriptomics to develop a CD8+ T cell specific gene signature for a productive anti-tumor response and show the response signature to be associated with overall survival (OS) on nivolumab monotherapy in CheckMate-067, a phase 3 clinical trial in metastatic melanoma. These results highlight the value of leveraging single-cell assays to dissect heterogeneous tumor and immune subsets and define cell-type specific transcriptomic biomarkers of ICB response.

Frontline Science: OX40 agonistic antibody reverses immune suppression and improves survival in sepsis.

A defining feature of protracted sepsis is development of immunosuppression that is thought to be a major driving force in the morbidity and mortality associated with the syndrome. The immunosuppression that occurs in sepsis is characterized by profound apoptosis-induced depletion of CD4 and CD8 T cells and severely impaired T cell function. OX40, a member of the TNF receptor superfamily, is a positive co-stimulatory molecule expressed on activated T cells. When engaged by OX40 ligand, OX40 stimulates T cell proliferation and shifts the cellular immune phenotype toward TH1 with increased production of cytokines that are essential for control of invading pathogens. The purpose of the present study was to determine if administration of agonistic Ab to OX40 could reverse sepsis-induced immunosuppression, restore T cell function, and improve survival in a clinically relevant animal model of sepsis. The present study demonstrates that OX40 agonistic Ab reversed sepsis-induced impairment of T cell function, increased T cell IFN-γ production, increased the number of immune effector cells, and improved survival in the mouse cecal ligation and puncture model of sepsis. Importantly, OX40 agonistic Ab was not only effective in murine sepsis but also improved T effector cell function in PBMCs from patients with sepsis. The present results provide support for the use of immune adjuvants that target T cell depletion and T cell dysfunction in the therapy of sepsis-induced immunosuppression. In addition to the checkpoint inhibitors anti-PD-1 and anti-PD-L1, OX40 agonistic Ab may be a new therapeutic approach to the treatment of this highly lethal disorder.

Safety and efficacy of anti-PD-L1 therapy in the woodchuck model of HBV infection.

Immune clearance of Hepatitis B virus (HBV) is characterized by broad and robust antiviral T cell responses, while virus-specific T cells in chronic hepatitis B (CHB) are rare and exhibit immune exhaustion that includes programmed-death-1 (PD-1) expression on virus-specific T cells. Thus, an immunotherapy able to expand and activate virus-specific T cells may have therapeutic benefit for CHB patients. Like HBV-infected patients, woodchucks infected with woodchuck hepatitis virus (WHV) can have increased hepatic expression of PD-1-ligand-1 (PD-L1), increased PD-1 on CD8+ T cells, and a limited number of virus-specific T cells with substantial individual variation in these parameters. We used woodchucks infected with WHV to assess the safety and efficacy of anti-PD-L1 monoclonal antibody therapy (αPD-L1) in a variety of WHV infection states. Experimentally-infected animals lacked PD-1 or PD-L1 upregulation compared to uninfected controls, and accordingly, αPD-L1 treatment in lab-infected animals had limited antiviral effects. In contrast, animals with naturally acquired WHV infections displayed elevated PD-1 and PD-L1. In these same animals, combination therapy with αPD-L1 and entecavir (ETV) improved control of viremia and antigenemia compared to ETV treatment alone, but with efficacy restricted to a minority of animals. Pre-treatment WHV surface antigen (sAg) level was identified as a statistically significant predictor of treatment response, while PD-1 expression on peripheral CD8+ T cells, T cell production of interferon gamma (IFN-γ) upon in vitro antigen stimulation (WHV ELISPOT), and circulating levels of liver enzymes were not. To further assess the safety of this strategy, αPD-L1 was tested in acute WHV infection to model the risk of liver damage when the extent of hepatic infection and antiviral immune responses were expected to be the greatest. No significant increase in serum markers of hepatic injury was observed over those in infected, untreated control animals. These data support a positive benefit/risk assessment for blockade of the PD-1:PD-L1 pathway in CHB patients and may help to identify patient groups most likely to benefit from treatment. Furthermore, the efficacy of αPD-L1 in only a minority of animals, as observed here, suggests that additional agents may be needed to achieve a more robust and consistent response leading to full sAg loss and durable responses through anti-sAg antibody seroconversion.

High-throughput screening and rapid inhibitor triage using an infectious chimeric Hepatitis C virus.

The recent development of a Hepatitis C virus (HCV) infectious virus cell culture model system has facilitated the development of whole-virus screening assays which can be used to interrogate the entire virus life cycle. Here, we describe the development of an HCV growth assay capable of identifying inhibitors against all stages of the virus life cycle with assay throughput suitable for rapid screening of large-scale chemical libraries. Novel features include, 1) the use of an efficiently-spreading, full-length, intergenotypic chimeric reporter virus with genotype 1 structural proteins, 2) a homogenous assay format compatible with miniaturization and automated liquid-handling, and 3) flexible assay end-points using either chemiluminescence (high-throughput screening) or Cellomics ArrayScan™ technology (high-content screening). The assay was validated using known HCV antivirals and through a large-scale, high-throughput screening campaign that identified novel and selective entry, replication and late-stage inhibitors. Selection and characterization of resistant viruses provided information regarding inhibitor target and mechanism. Leveraging results from this robust whole-virus assay represents a critical first step towards identifying inhibitors of novel targets to broaden the spectrum of antivirals for the treatment of HCV.

A novel small molecule inhibitor of hepatitis C virus entry.

Small molecule inhibitors of hepatitis C virus (HCV) are being developed to complement or replace treatments with pegylated interferons and ribavirin, which have poor response rates and significant side effects. Resistance to these inhibitors emerges rapidly in the clinic, suggesting that successful therapy will involve combination therapy with multiple inhibitors of different targets. The entry process of HCV into hepatocytes represents another series of potential targets for therapeutic intervention, involving viral structural proteins that have not been extensively explored due to experimental limitations. To discover HCV entry inhibitors, we utilized HCV pseudoparticles (HCVpp) incorporating E1-E2 envelope proteins from a genotype 1b clinical isolate. Screening of a small molecule library identified a potent HCV-specific triazine inhibitor, EI-1. A series of HCVpp with E1-E2 sequences from various HCV isolates was used to show activity against all genotype 1a and 1b HCVpp tested, with median EC50 values of 0.134 and 0.027 µM, respectively. Time-of-addition experiments demonstrated a block in HCVpp entry, downstream of initial attachment to the cell surface, and prior to or concomitant with bafilomycin inhibition of endosomal acidification. EI-1 was equally active against cell-culture adapted HCV (HCVcc), blocking both cell-free entry and cell-to-cell transmission of virus. HCVcc with high-level resistance to EI-1 was selected by sequential passage in the presence of inhibitor, and resistance was shown to be conferred by changes to residue 719 in the carboxy-terminal transmembrane anchor region of E2, implicating this envelope protein in EI-1 susceptibility. Combinations of EI-1 with interferon, or inhibitors of NS3 or NS5A, resulted in additive to synergistic activity. These results suggest that inhibitors of HCV entry could be added to replication inhibitors and interferons already in development.

Genotypic determinants and phenotypic properties of antiviral-resistant HBV variants: insight from entecavir resistance studies.

Successful antiviral therapy results in cessation of disease progression and reversal of symptoms, and often can eliminate the virus infection. The emergence of virus with resistance to therapy, however, can abrogate these benefits; therefore, it is important to determine the pathways and frequency of resistance to antivirals. Often, resistance pathways can be elucidated in preclinical experiments through the isolation and characterization of resistant variants in cell culture or animals. For viruses without adequate cell culture systems, however, resistance pathways must await identification through analysis of isolates from patients failing therapy. Such is the case for antivirals used to treat chronic hepatitis B. Here, we detail the process used to define the pathways and frequency of resistance using genotypic and phenotypic assays during the development of entecavir, a novel therapy for chronic HBV infection. The scheme takes into account the need for rapid analysis of large numbers of clinical isolates through nucleotide sequencing and methods for phenotypic validation using cell culture and in vitro enzyme immunoassay formats.

Mechanistic characterization and molecular modeling of hepatitis B virus polymerase resistance to entecavir.

BACKGROUND: Entecavir (ETV) is a deoxyguanosine analog competitive inhibitor of hepatitis B virus (HBV) polymerase that exhibits delayed chain termination of HBV DNA. A high barrier to entecavir-resistance (ETVr) is observed clinically, likely due to its potency and a requirement for multiple resistance changes to overcome suppression. Changes in the HBV polymerase reverse-transcriptase (RT) domain involve lamivudine-resistance (LVDr) substitutions in the conserved YMDD motif (M204V/I +/- L180M), plus an additional ETV-specific change at residues T184, S202 or M250. These substitutions surround the putative dNTP binding site or primer grip regions of the HBV RT. METHODS/PRINCIPAL FINDINGS: To determine the mechanistic basis for ETVr, wildtype, lamivudine-resistant (M204V, L180M) and ETVr HBVs were studied using in vitro RT enzyme and cell culture assays, as well as molecular modeling. Resistance substitutions significantly reduced ETV incorporation and chain termination in HBV DNA and increased the ETV-TP inhibition constant (K(i)) for HBV RT. Resistant HBVs exhibited impaired replication in culture and reduced enzyme activity (k(cat)) in vitro. Molecular modeling of the HBV RT suggested that ETVr residue T184 was adjacent to and stabilized S202 within the LVDr YMDD loop. ETVr arose through steric changes at T184 or S202 or by disruption of hydrogen-bonding between the two, both of which repositioned the loop and reduced the ETV-triphosphate (ETV-TP) binding pocket. In contrast to T184 and S202 changes, ETVr at primer grip residue M250 was observed during RNA-directed DNA synthesis only. Experimentally, M250 changes also impacted the dNTP-binding site. Modeling suggested a novel mechanism for M250 resistance, whereby repositioning of the primer-template component of the dNTP-binding site shifted the ETV-TP binding pocket. No structural data are available to confirm the HBV RT modeling, however, results were consistent with phenotypic analysis of comprehensive substitutions of each ETVr position. CONCLUSIONS: Altogether, ETVr occurred through exclusion of ETV-TP from the dNTP-binding site, through different, novel mechanisms that involved lamivudine-resistance, ETV-specific substitutions, and the primer-template.

Entecavir treatment for up to 5 years in patients with hepatitis B e antigen-positive chronic hepatitis B.

UNLABELLED: Sustained virologic suppression is a primary goal of therapy for chronic hepatitis B (CHB). In study entecavir (ETV)-022, 48 weeks of entecavir 0.5 mg was superior to lamivudine for virologic suppression for hepatitis B e antigen (HBeAg)-positive CHB. A total of 183 entecavir-treated patients from ETV-022 subsequently enrolled in study ETV-901. We present the results after up to 5 years (240 weeks) of continuous entecavir therapy. The entecavir long-term cohort consists of patients who received >or=1 year of entecavir 0.5 mg in ETV-022 and then entered ETV-901 with a treatment gap

Two cases of development of entecavir resistance during entecavir treatment for nucleoside-naive chronic hepatitis B.

BACKGROUND: Entecavir (ETV) is a potent nucleoside analogue against hepatitis B virus (HBV), and emergence of drug resistance is rare in nucleoside-naive patients because development of ETV resistance (ETVr) requires at least three amino acid substitutions in HBV reverse transcriptase. We observed two cases of genotypic ETVr with viral rebound and biochemical breakthrough during ETV treatment of nucleoside-naive patients with chronic hepatitis B (CHB). RESULTS: Case 1: A 44-year-old HBeAg-positive man received ETV 0.1 mg/day for 52 weeks and 0.5 mg/day for 96 weeks consecutively. HBV DNA was 10.0 log(10) copies/ml at baseline, declined to a nadir of 3.1 at week 100, and rebounded to 4.5 at week 124 and 6.7 at week 148. Alanine aminotransferase (ALT) level increased to 112 IU/l at week 148. Switching to a lamivudine (LVD)/adefovir-dipivoxil combination was effective in decreasing HBV DNA. Case 2: A 47-year-old HBeAg-positive man received ETV 0.5 mg/day for 188 weeks. HBV DNA was 8.2 log(10) copies/ml at baseline, declined to a nadir of 2.9 at week 124, and then rebounded to 4.7 at week 148 and 6.4 at week 160. ALT level increased to 72 IU/l at week 172. The ETVr-related substitution (S202G), along with LVD-resistance-related substitutions (L180M and M204V), was detected by sequence analysis at week 124 in both case 1 and case 2. CONCLUSIONS: ETVr emerged in two Japanese nucleoside-naive CHB patients after prolonged therapy and incomplete suppression and in one patient after <0.5 mg of dosing. ETV patients with detectable HBV DNA or breakthrough after extended therapy should be evaluated for compliance to therapy and potential emergence of resistance.

Ultrasensitive genotypic detection of antiviral resistance in hepatitis B virus clinical isolates.

Amino acid substitutions that confer reduced susceptibility to antivirals arise spontaneously through error-prone viral polymerases and are selected as a result of antiviral therapy. Resistance substitutions first emerge in a fraction of the circulating virus population, below the limit of detection by nucleotide sequencing of either the population or limited sets of cloned isolates. These variants can expand under drug pressure to dominate the circulating virus population. To enhance detection of these viruses in clinical samples, we established a highly sensitive quantitative, real-time allele-specific PCR assay for hepatitis B virus (HBV) DNA. Sensitivity was accomplished using a high-fidelity DNA polymerase and oligonucleotide primers containing locked nucleic acid bases. Quantitative measurement of resistant and wild-type variants was accomplished using sequence-matched standards. Detection methodology that was not reliant on hybridization probes, and assay modifications, minimized the effect of patient-specific sequence polymorphisms. The method was validated using samples from patients chronically infected with HBV through parallel sequencing of large numbers of cloned isolates. Viruses with resistance to lamivudine and other l-nucleoside analogs and entecavir, involving 17 different nucleotide substitutions, were reliably detected at levels at or below 0.1% of the total population. The method worked across HBV genotypes. Longitudinal analysis of patient samples showed earlier emergence of resistance on therapy than was seen with sequencing methodologies, including some cases of resistance that existed prior to treatment. In summary, we established and validated an ultrasensitive method for measuring resistant HBV variants in clinical specimens, which enabled earlier, quantitative measurement of resistance to therapy.

Long-term monitoring shows hepatitis B virus resistance to entecavir in nucleoside-naïve patients is rare through 5 years of therapy.

UNLABELLED: Patients with chronic hepatitis B virus (HBV) infection who develop antiviral resistance lose benefits of therapy and may be predisposed to further resistance. Entecavir (ETV) resistance (ETVr) results from HBV reverse transcriptase substitutions at positions T184, S202, or M250, which emerge in the presence of lamivudine (LVD) resistance substitutions M204I/V +/- L180M. Here, we summarize results from comprehensive resistance monitoring of patients with HBV who were continuously treated with ETV for up to 5 years. Monitoring included genotypic analysis of isolates from all patients at baseline and when HBV DNA was detectable by polymerase chain reaction (> or = 300 copies/mL) from Years 1 through 5. In addition, genotyping was performed on isolates from patients experiencing virologic breakthrough (> or = 1 log(10) rise in HBV DNA). In vitro phenotypic ETV susceptibility was determined for virologic breakthrough isolates, and for HBV containing novel substitutions emerging during treatment. The results over 5 years of therapy showed that in nucleoside-naïve patients, the cumulative probability of genotypic ETVr and genotypic ETVr associated with virologic breakthrough was 1.2% and 0.8%, respectively. In contrast, a reduced barrier to resistance was observed in LVD-refractory patients, as the LVD resistance substitutions, a partial requirement for ETVr, preexist, resulting in a 5-year cumulative probability of genotypic ETVr and genotypic ETVr associated with breakthrough of 51% and 43%, respectively. Importantly, only four patients who achieved < 300 copies/mL HBV DNA subsequently developed ETVr. CONCLUSION: Long-term monitoring showed low rates of resistance in nucleoside-naïve patients during 5 years of ETV therapy, corresponding with potent viral suppression and a high genetic barrier to resistance. These findings support ETV as a primary therapy that enables prolonged treatment with potent viral suppression and minimal resistance.

Entecavir therapy for lamivudine-refractory chronic hepatitis B: improved virologic, biochemical, and serology outcomes through 96 weeks.

UNLABELLED: In hepatitis B e antigen (HBeAg)-positive chronic hepatitis B patients who were refractory to current lamivudine therapy, switching to entecavir was superior to continued lamivudine at week 48 for histologic improvement, viral load reduction by polymerase chain reaction and alanine aminotransferase normalization. We assessed the efficacy, safety, and resistance profile of entecavir through 96 weeks of treatment. A total of 286 patients were randomized and treated with entecavir 1 mg (n = 141) or continued lamivudine 100 mg (n = 145). At week 52, 77 entecavir-treated patients who had a protocol-defined virologic response (HBV branched DNA [bDNA] < 0.7 MEq/mL but HBeAg-positive) continued blinded therapy for up to 96 weeks. Patients were assessed for efficacy, safety, and emerging resistance. Cumulative proportions of all treated patients who achieved confirmed efficacy endpoints were also analyzed. Between week 48 and the end of dosing, the proportions of patients with HBV DNA <300 copies/mL by polymerase chain reaction increased from 21% to 40%, and alanine aminotransferase normalization (< or =1x upper limit of normal) increased from 65% to 81%. In the second year, HBeAg seroconversion was achieved by 10% of patients. Of the 77 patients in the second year treatment cohort, entecavir resistance emerged in six patients, and seven experienced virologic breakthrough (five with genotypic resistance acquired before year 2). The safety profile of entecavir in the second year of therapy was consistent with that reported during year 1. CONCLUSION: Through 96 weeks of treatment, 1 mg entecavir resulted in continued clinical benefit in lamivudine-refractory HBeAg-positive chronic hepatitis B patients with a safety profile comparable to lamivudine.

Comprehensive evaluation of hepatitis B virus reverse transcriptase substitutions associated with entecavir resistance.

UNLABELLED: Virologic resistance emerging during entecavir (ETV) therapy for hepatitis B virus (HBV) requires three substitutions in the viral reverse transcriptase (RT), signifying a high barrier to resistance. Two of these substitutions are associated with lamivudine resistance (LVDr) in the tyrosine-methionine-aspartate-aspartate (YMDD) motif (rtM204V and rtL180M), whereas the other occurs at one or more positions specifically associated with ETV resistance (ETVr): rtT184, rtS202, or rtM250. Although a variety of substitutions at these primary ETVr positions arise during ETV therapy, only a subset give rise to clinical virologic breakthrough. To determine the phenotypic impact of observed clinical and potential new ETVr substitutions, a comprehensive panel of clones containing every possible amino acid at the three primary ETVr positions in LVDr HBV was constructed and analyzed in vitro. A range of replication capacities was observed for the panel, but none of the mutations rescued replication of the LVDr mutant to the wild-type level. More clones with residue rtS202 substitutions were severely impaired than those at rtT184 or rtM250. A wide variety of ETV susceptibilities was observed, ranging from approximately eight-fold (no increase over the LVDr parent) to greater than 400-fold over the wild-type. A correlation was identified between clinically observed substitutions and those displaying higher in vitro replication and resistance, especially those from virologic breakthrough patients. CONCLUSION: The high number of tolerated and resistant ETVr substitutions is consistent with models predicting that the mechanism for ETVr is through enhancement of LVDr changes in the RT deoxyribonucleotide triphosphate (dNTP)-binding pocket.

Hepatitis B virus quasispecies susceptibility to entecavir confirms the relationship between genotypic resistance and patient virologic response.

BACKGROUND/AIMS: The efficacy of anti-viral therapy for chronic hepatitis B virus (HBV) is lost upon the emergence of resistant virus. Using >500 patient HBV isolates from several entecavir clinical trials, we show that phenotypic susceptibility correlates with genotypic resistance and patient virologic responses. METHODS: The full-length HBV or reverse transcriptase gene was amplified from patient sera, sequenced, and cloned into an HBV expression vector. Entecavir susceptibilities of individual virus clones and patient quasispecies populations were analyzed in conjunction with the sequenced resistance genotype and the patient's virologic response. RESULTS: Entecavir susceptibility decreased approximately 8-fold for isolates with various constellations of lamivudine resistance substitutions. The spectrum of additional substitutions that emerged during therapy at residues rtT184, rtS202, or rtM250 displayed varying levels of entecavir susceptibility according to the specific resistance substitutions and the proportion of resistant variants in the quasispecies. Phenotypic analyses of samples associated with virologic breakthrough confirmed the role of these residue changes in entecavir resistance. Additional longitudinal phenotypic analyses showed that decreased susceptibility correlated with both genotypic resistance and increased circulating HBV DNA. CONCLUSIONS: HBV phenotypic analysis provides additional insight as part of a resistance monitoring program that includes genotypic analysis and quantification of circulating virus.

Entecavir for treatment of hepatitis B virus displays no in vitro mitochondrial toxicity or DNA polymerase gamma inhibition.

Therapy with nucleoside reverse transcriptase inhibitors (NRTIs) can be associated with mitochondrial toxicity. In vitro studies have been used to predict the predisposition for and characterize the mechanisms causing mitochondrial toxicity. Entecavir (ETV) is an approved deoxyguanosine nucleoside for the treatment of chronic hepatitis B virus (HBV) infection that exhibits potent activity against viral reverse transcriptase. We assessed the potential for mitochondrial toxicity of ETV in long-term cultures of HepG2 hepatoma cells by measuring mitochondrial function (through lactate secretion), levels of mitochondrial DNA (mtDNA), and levels of mitochondrial proteins COX II and COX IV. Furthermore, we tested the activity of ETV-triphosphate (ETV-TP) against mitochondrial DNA polymerase gamma (Pol gamma) in vitro. ETV concentrations as high as 100 times the maximal clinical exposure (C(max)) did not affect cell proliferation, levels of lactate, mitochondrial DNA, or mitochondrial proteins throughout the 15-day culture. The lack of mitochondrial toxicity was consistent with the finding that ETV-TP was not recognized by mitochondrial DNA Pol gamma and failed to be incorporated into DNA or inhibit the polymerase assay at the highest levels tested, 300 microM. Combinations of ETV with each of the other HBV NRTI antivirals, adefovir, tenofovir, and lamivudine at 10 times their respective C(max) levels also failed to result in cellular or mitochondrial toxicity. In summary, cell culture and enzymatic studies yielded no evidence that would predict mitochondrial toxicity of ETV at exposure levels in excess of those expected to be achieved clinically.

Inhibition of hepatitis B virus polymerase by entecavir.

Entecavir (ETV; Baraclude) is a novel deoxyguanosine analog with activity against hepatitis B virus (HBV). ETV differs from the other nucleoside/tide reverse transcriptase inhibitors approved for HBV therapy, lamivudine (LVD) and adefovir (ADV), in several ways: ETV is >100-fold more potent against HBV in culture and, at concentrations below 1 microM, displays no significant activity against human immunodeficiency virus (HIV). Additionally, while LVD and ADV are obligate DNA chain terminators, ETV halts HBV DNA elongation after incorporating a few additional bases. Three-dimensional homology models of the catalytic center of the HBV reverse transcriptase (RT)-DNA-deoxynucleoside triphosphate (dNTP) complex, based on the HIV RT-DNA structure, were used with in vitro enzyme kinetic studies to examine the mechanism of action of ETV against HBV RT. A novel hydrophobic pocket in the rear of the RT dNTP binding site that accommodates the exocyclic alkene moiety of ETV was predicted, establishing a basis for the superior potency observed experimentally. HBV DNA chain termination by ETV was accomplished through disfavored energy requirements as well as steric constraints during subsequent nucleotide addition. Validation of the model was accomplished through modeling of LVD resistance substitutions, which caused an eightfold decrease in ETV susceptibility and were predicted to reduce, but not eliminate, the ETV-binding pocket, in agreement with experimental observations. ADV resistance changes did not affect the ETV docking model, also agreeing with experimental results. Overall, these studies explain the potency, mechanism, and cross-resistance profile of ETV against HBV and account for the successful treatment of naive and LVD- or ADV-experienced chronic HBV patients.

Two-year assessment of entecavir resistance in Lamivudine-refractory hepatitis B virus patients reveals different clinical outcomes depending on the resistance substitutions present.

Entecavir (ETV) is a deoxyguanosine analog approved for use for the treatment of chronic infection with wild-type and lamivudine-resistant (LVDr) hepatitis B virus (HBV). In LVD-refractory patients, 1.0 mg ETV suppressed HBV DNA levels to below the level of detection by PCR (<300 copies/ml) in 21% and 34% of patients by Weeks 48 and 96, respectively. Prior studies showed that virologic rebound due to ETV resistance (ETVr) required preexisting LVDr HBV reverse transcriptase substitutions M204V and L180M plus additional changes at T184, S202, or M250. To monitor for resistance, available isolates from 192 ETV-treated patients were sequenced, with phenotyping performed for all isolates with all emerging substitutions, in addition to isolates from all patients experiencing virologic rebounds. The T184, S202, or M250 substitution was found in LVDr HBV at baseline in 6% of patients and emerged in isolates from another 11/187 (6%) and 12/151 (8%) ETV-treated patients by Weeks 48 and 96, respectively. However, use of a more sensitive PCR assay detected many of the emerging changes at baseline, suggesting that they originated during LVD therapy. Only a subset of the changes in ETVr isolates altered their susceptibilities, and virtually all isolates were significantly replication impaired in vitro. Consequently, only 2/187 (1%) patients experienced ETVr rebounds in year 1, with an additional 14/151 (9%) patients experiencing ETVr rebounds in year 2. Isolates from all 16 patients with rebounds were LVDr and harbored the T184 and/or S202 change. Seventeen other novel substitutions emerged during ETV therapy, but none reduced the susceptibility to ETV or resulted in a rebound. In summary, ETV was effective in LVD-refractory patients, with resistant sequences arising from a subset of patients harboring preexisting LVDr/ETVr variants and with approximately half of the patients experiencing a virologic rebound.